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(A) Mass spectrometry analysis of NLRP3, <t>RACK1,</t> and NEK7 peptides after purification of NLRP3 complexes.
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(A) Mass spectrometry analysis of NLRP3, RACK1, and NEK7 peptides after purification of NLRP3 complexes.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Mass spectrometry analysis of NLRP3, RACK1, and NEK7 peptides after purification of NLRP3 complexes.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Mass Spectrometry, Purification

(A–C) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). The cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h), ATP (5 mM, 30 min), or LPS (200 ng mL−1, 4 h) plus ATP (5 mM, 30 min). (A) Supernatants (Sup.) and cell lysates (Lys.) were analyzed by immunoblotting with the indicated antibodies. (B and C) IL-1β and TNF-α release in supernatants was analyzed by ELISA.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A–C) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). The cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h), ATP (5 mM, 30 min), or LPS (200 ng mL−1, 4 h) plus ATP (5 mM, 30 min). (A) Supernatants (Sup.) and cell lysates (Lys.) were analyzed by immunoblotting with the indicated antibodies. (B and C) IL-1β and TNF-α release in supernatants was analyzed by ELISA.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

(A–E) Macrophages (NLRP3 R258W) were treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h). Cell lysates (Lys.) and culture supernatants (Sup.) were immunoblotted with indicated antibodies (A and D). IL-1β (B), TNF-α (C), and LDH(E) in culture supernatants of macrophages were analyzed. Error bars denote SD of triplicate wells. Results are representative of three independent experiments. Unpaired two-tailed Student’s t test, *p < 0.05.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A–E) Macrophages (NLRP3 R258W) were treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were left unstimulated (PBS) or stimulated with LPS (200 ng mL−1, 4 h). Cell lysates (Lys.) and culture supernatants (Sup.) were immunoblotted with indicated antibodies (A and D). IL-1β (B), TNF-α (C), and LDH(E) in culture supernatants of macrophages were analyzed. Error bars denote SD of triplicate wells. Results are representative of three independent experiments. Unpaired two-tailed Student’s t test, *p < 0.05.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Two Tailed Test

(A) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were primed with LPS (200 ng mL−1, 4 h) and then stimulated with ATP (5 mM, 30 min), nigericin (5 μM, 1 h), or Salmonella (MOI = 10, 1 h). Representative images of ASC specks in macrophages treated with indicated stimuli. Scale bars, 5 μm.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) iBMDM was treated with control siRNA (siControl) or RACK1 siRNA (siRACK1). Cells were primed with LPS (200 ng mL−1, 4 h) and then stimulated with ATP (5 mM, 30 min), nigericin (5 μM, 1 h), or Salmonella (MOI = 10, 1 h). Representative images of ASC specks in macrophages treated with indicated stimuli. Scale bars, 5 μm.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques:

(A) Tagged NLRP3 (NLRP3-SFP) was co-expressed with HA-tagged wild-type RACK1 or mutant RACK1 (R36D/K38E, defective in ribosome binding) in HEK293T cells. Cell lysates were pulled down with streptavidin beads and analyzed by immunoblotting.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Tagged NLRP3 (NLRP3-SFP) was co-expressed with HA-tagged wild-type RACK1 or mutant RACK1 (R36D/K38E, defective in ribosome binding) in HEK293T cells. Cell lysates were pulled down with streptavidin beads and analyzed by immunoblotting.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Mutagenesis, Binding Assay, Western Blot

(A) Macrophages were infected with lentivirus expressing control shRNA (shControl) or RACK1 shRNA (shRACK1). After puromycin selection, transduced macrophages were primed with LPS (200 ng mL−1, 4 h) and then stimulated with PBS (control), ATP (5 mM, 30 min), or nigericin (5 μM, 1 h). NLRP3 inflammasome assembly was analyzed by blue native PAGE and immunoblotting. Cell lysates were also analyzed by SDS–PAGE and immunoblotting.

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: (A) Macrophages were infected with lentivirus expressing control shRNA (shControl) or RACK1 shRNA (shRACK1). After puromycin selection, transduced macrophages were primed with LPS (200 ng mL−1, 4 h) and then stimulated with PBS (control), ATP (5 mM, 30 min), or nigericin (5 μM, 1 h). NLRP3 inflammasome assembly was analyzed by blue native PAGE and immunoblotting. Cell lysates were also analyzed by SDS–PAGE and immunoblotting.

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Infection, Expressing, shRNA, Selection, Blue Native PAGE, Western Blot, SDS Page

KEY RESOURCES TABLE

Journal: Cell reports

Article Title: RACK1 Mediates NLRP3 Inflammasome Activation by Promoting NLRP3 Active Conformation and Inflammasome Assembly

doi: 10.1016/j.celrep.2020.108405

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: This work was funded in part by National Institutes of Health grants (R01AI148544 to Y.H, R01AI06331 to G.N.), Wayne State startup funds (Y.H), a Ministerio de Economía, Industria y Competitividad grant (SAF2017-88276-Rto P.P.), a Fundación Séneca grant (20859/PI/18 to P.P), and a European Research Council grant (ERC-2013-CoG 614578 to P.P.). pEGFP-N1-RACK1 (Addgene plasmid 41088) was a gift from Anna Huttenlocher. pHIV-EGFP (Addgene plasmid 21373) was a gift from Bryan Welm and Zena Werb. pCMV-dR8.2 dvpr (Addgene plasmid 8455), and pCMVVSV-G (Addgene plasmid 8454) were gifts from Bob Weinberg.

Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Mutagenesis, In Vivo, Plasmid Preparation, Software